High-activity pectinase DS-5 as well as preparation and application thereof

Abstract

The invention discloses high-activity pectinase DS-5 as well as preparation and application thereof, and is characterized in that a deep learning model ProteinMPNN is utilized to redesign a wild type pectinase sequence, and a pectinase mutant DS-5 of which the enzyme activity is improved by more than 8 times compared with that of the wild type is obtained. Compared with a WT sequence, 72 amino acids in total are replaced. The high-activity pectinase DS-5 disclosed by the invention has a wide application prospect in the industrial fields of food processing, spinning, papermaking, biological energy sources and the like.

Description

Technical Field

[0001] The present invention relates to the fields of bioengineering and enzyme engineering, and in particular to a highly active pectinase mutant DS-5 designed by a deep learning model ProteinMPNN. Background Art

[0002] Pectinase is a type of enzyme that can hydrolyze pectin and has important application value in food processing, textile, papermaking, bioenergy and other industrial fields. Improving the activity and stability of pectinase is of great significance for industrial production.

[0003] Traditional enzyme engineering methods such as directed evolution and rational design have achieved certain results in improving enzyme performance, but they have limitations such as long time consumption, low efficiency, and limited exploration of sequence space. With the development of deep learning technology, the design and reconstruction of proteins using deep learning models has become an emerging trend. ProteinMPNN is a protein sequence design model based on deep learning that can generate sequences with optimized functions while maintaining the tertiary structure of proteins. Summary of the invention

[0004] The purpose of the present invention is to provide a high-activity pectinase DS-5, the enzyme activity of which is increased eight times compared with wild-type pectinase (WT).

[0005] The objective of the present invention is achieved through the following technical solutions: A highly active pectinase DS-5, whose amino acid sequence is shown in SEQ ID NO: 2, is obtained by redesigning the wild-type pectinase sequence using a deep learning model ProteinMPNN, and screening out a highly active pectinase mutant DS-5.

[0006] The specific technical solutions are as follows: 1. Acquisition of DS-5 mutants: o The protein MPNN model was used to redesign the pectinase sequence using the crystal structure of wild-type pectinase (PDB ID: 3VMW) as input.

[0007] o Based on multiple sequence alignment (MSA), approximately 70% of the highly conserved residues were kept unchanged, and sequence variation design was performed on the remaining approximately 30% of non-conserved residues.

[0008] o Among the 16 variant sequences generated by the model, a highly active mutant DS-5 was screened.

[0009] 2. The different amino acid sites between DS-5 and WT sequences: o WT pectinase sequence (SEQ ID NO: 1): GPQGYASMNGGTTGGAGGRVEYASTGAQIQQLIDNRSRSNNPDEPLTIYVNGTITQGNSPQSLIDVKNHRGKAHEIKNISIIGVGTNGEFDGIGIRLSNAHNIIIQNVSIHHVREGEGTAIEVTDDSKNVWIDHNEFYSEFPGNGDSDYYDGLVDMKRNAEYITVSWNKFENHWKTMLVGHTDNASLAPDKITYHHNYFNNLNSRVPLIRYADVHMFNNYFKDINDTAINSRVGARVFVENNYFDNVGSGQADPTTGFIKGPVGWFYGSPSTGYWNLRGNVFVNTPNSHLNSTTNFTPPYSYQVQSATQAKSSVEQHSGVGVIN o DS-5 pectinase sequence (SEQ ID NO: 2): GPQGYASLNGGTTGGSGGDTATASTGKEIQDLIDTRSLSSNPNKPLTIYVNGTITPANSDKPLIDVKNHRGSEYPITNISIIGVGTNGEFDGIGIKLSNAHNIIIQNVKIHHVKEGTGTCIEVTDNSKNVWIDHNEFYSDYPGNGDSDYYDGLVDIKRNAEYITVSWNKFENHWKAMLVGHTDNPALAPDNITYHHNLFKNLNSRVPLIRYANVHMFNNYFEDIKDTAINSRVGARVWVENNYFDNVGSGKKDPVTGEVRGPVGHYYGSSSKGYWNLKGNIFNNTPHSGLKSTTDFEPPYSYTVLSAEEAKAYVLKNSGVGVKT o Information on differential amino acid sites: By comparing the amino acid sequences of WT and DS-5, it was found that a total of 72 amino acid sites in DS-5 were replaced. The main mutation sites are listed below (sequence positions refer to WT sequence): M8L, A16S, R19D, V20T, E21A, Y22T,A27K, Q28E, Q31D, N35T, R38L, N40S, D43N, E44K, Q56P, G57A, P60D, Q61K, S62P,K72S, A73E, H74Y,E75P, K77T, R96K, S109K, R114K, A120C, D126N, E140D,F141Y, M156I, T176A, A185P, S186A, K191N, Y198L, N200K, D213N, K222E, N225K,F238W, Q251K,A252K, T255V, F258E, I259V, K260R, W265H, F266Y, P270S, T272K,R278K, V281I, V283N, N287H, H289G, N291K, N295D, T297E, Q303T, Q305L, T308E, Q309E, S312A, S313Y, E315L, Q316K, H317N, I323K, N324T.

[0010] 3. Preparation method of DS-5: o Based on the amino acid sequence of DS-5, the corresponding gene sequence was synthesized using gene synthesis technology.

[0011] o Clone the synthesized gene sequence into an appropriate expression vector.

[0012] o Transform into suitable host bacteria (such as Escherichia coli, Bacillus) for expression.

[0013] o Through fermentation culture, the high-activity DS-5 pectinase was induced to express.

[0014] o Use conventional protein purification methods (such as ion exchange, affinity chromatography, etc.) to purify DS-5 pectinase.

[0015] 4. Application of DS-5: Highly active pectinase DS-5 can be widely used in the following fields: Food processing: used for juice clarification, jam production, etc. to improve product quality and production efficiency.

[0016] Textile industry: used in bio-enzyme desizing and bio-polishing to improve the quality of textiles.

[0017] Papermaking industry: used for biological bleaching of pulp, reducing the use of chemical reagents and reducing environmental pollution.

[0018] Bioenergy: Participate in biomass degradation, improve raw material conversion efficiency, and promote bioenergy production.

[0019] The biggest feature of the present invention is that the pectinase sequence is redesigned by using the deep learning model ProteinMPNN, and the pectinase mutant DS-5 is obtained, whose enzyme activity is more than 8 times higher than that of the wild type. Compared with the WT sequence, DS-5 has a total of 72 amino acids replaced. The pectinase has broad application prospects in industrial fields such as food processing, textiles, papermaking and bioenergy. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : The results of enzyme activity assay showed that the catalytic activity of DS-5 was significantly higher than that of WT (wild type).

[0021] Figure 2 : SDS-PAGE electrophoresis of WT and DS-5 purified proteins to evaluate the purity and molecular weight of the proteins.

[0022] Figure 3 : Electrostatic surface potential diagram of WT and DS-5 proteins (WT on the left, DS-5 on the right). The black dotted circle outlines the potential substrate binding pocket, showing that DS-5 has an increased positive potential in this area. DETAILED DESCRIPTION

[0023] The present invention is further described in detail below in conjunction with the embodiments (attached drawings): Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; unless otherwise specified, the materials used are all available from commercial sources.

[0024] Example 1: Construction of DS-5 gene In order to obtain the highly active pectinase mutant DS-5, it is first necessary to obtain its optimized amino acid sequence. During the research, the deep learning algorithm ProteinMPNN was used to redesign the amino acid sequence of the enzyme using the crystal structure of pectinase (PDB number: 3VMW) as input. The highly conserved amino acid sites in pectinase were determined by multiple sequence alignment (MSA), 70% of the conserved residues were fixed, and the remaining 30% of the residues with large variations were sequenced. The ProteinMPNN algorithm generated 32 unique sequences. After structure prediction and quality assessment by AlphaFold2, 16 candidate sequences were screened out, and the mutant DS-5 with significantly improved activity was found through activity identification.

[0025] According to the amino acid sequence of DS-5, the reverse translation method was used to reversely infer the corresponding nucleotide sequence using the codons optimized by E. coli to ensure efficient expression in the prokaryotic expression system. Taking into account factors such as codon usage preference and mRNA secondary structure, the nucleotide sequence was optimized to reduce the use of rare codons and reduce possible obstacles in the transcription and translation process.

[0026] The optimized DS-5 gene sequence was synthesized and cloned into the prokaryotic expression vector pET-28a(+) to construct the recombinant expression vector pET-28a(+)-DS-5. The specific steps are as follows: 1. Enzyme digestion and ligation: Use restriction endonucleases NcoI and XhoI to digest the vector and DS-5 gene fragment.

[0027] 2. Ligation reaction: The DS-5 gene fragment after restriction digestion was connected with the linearized pET-28a(+) vector under the action of T4 DNA ligase to construct a recombinant plasmid.

[0028] 3. Transformation and screening: The ligation product was transformed into E. coli DH5α competent cells, spread on LB plates containing kanamycin, and cultured at 37°C overnight.

[0029] 4. Identification of positive clones: Pick a single colony for bacterial liquid PCR and enzyme digestion identification to screen out the correct recombinant plasmid.

[0030] 5. Sequencing verification: Send the screened recombinant plasmid to a sequencing company for sequence determination to confirm the correctness and integrity of the DS-5 gene sequence. Example 2: Expression and purification of DS-5 1. Strain transformation: The sequencing-verified recombinant plasmid pET-28a(+)-DS-5 was transformed into Escherichia coli BL21 (DE3) competent cells using the heat shock method, followed by shaking recovery in SOC medium without antibiotics for 1 hour.

[0032] 2. Preliminary culture: Spread the revived bacterial solution on an LB plate containing 50 μg / mL kanamycin and culture at 37°C overnight to obtain a single colony. Pick a single colony and inoculate it into 5 mL of LB liquid culture medium containing kanamycin and culture it at 37°C and 200 rpm overnight.

[0033] 3. Scale culture and induced expression: Inoculate the overnight cultured bacteria into fresh LB medium at a ratio of 1:100, and culture at 37°C and 200 rpm until the OD600 of the bacteria reaches 0.6-0.8. At this time, lower the culture temperature to 16°C, add isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.5 mM to induce expression, and continue to culture for 12-16 hours. A lower induction temperature helps to improve the solubility of the recombinant protein.

[0034] 4. Bacterial cell collection and lysis: After the culture is completed, the cells are collected by centrifugation at 4°C and 8000 rpm for 10 minutes. The cells are resuspended in pre-cooled lysis buffer (50 mM Tris-HCl, 300 mM NaCl, pH 8.0), protease inhibitors are added, and the cells are disrupted using an ultrasonic cell disruptor under ice bath conditions (working for 3 seconds, interval of 5 seconds, and cycle for 30 minutes).

[0035] 5. Obtaining crude enzyme solution: Centrifuge the lysed bacterial solution at 12,000 rpm and 4°C for 30 minutes, and collect the supernatant as the crude enzyme solution.

[0036] 6. Protein purification: Utilize the 6×His tag at the N-terminus of the recombinant protein and use a nickel ion affinity chromatography column (Ni-NTA) to purify the protein. The steps are as follows: Equilibration: Equilibrate the Ni-NTA column with binding buffer (50 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0).

[0037] Loading: Load the crude enzyme solution onto the equilibrated affinity column at an appropriate flow rate to allow the protein to fully bind.

[0038] Elution: The protein was eluted with different concentrations of imidazole eluent (40 mM, 80 mM, 200 mM imidazole) and the eluted fractions were collected.

[0039] Protein identification: SDS-PAGE was used to analyze each fraction to confirm the presence of the target protein.

[0040] 7. Protein concentration and dialysis: The fraction containing the target protein was concentrated by ultrafiltration and dialyzed overnight in a dialysis bag at 4°C to remove excess salt and imidazole.

[0041] Purity test: The purity of the purified DS-5 protein was tested by SDS-PAGE gel electrophoresis, and the results showed a single band at about 40 kDa (see Figure 2 ), indicating that the protein has high purity and can be used for subsequent experiments. Example 3: Determination of enzyme activity 1. Substrate preparation: Prepare a 0.5% (w / v) solution of citrus pectin in 0.05 M citric acid-sodium citrate buffer (pH 4.8) as the substrate for the enzyme reaction.

[0043] 2. Enzyme reaction system: Add 900 μL of substrate solution to a test tube and preheat to 37°C. Then add 100 μL of appropriately diluted DS-5 or WT enzyme solution, mix well, and react in a 37°C water bath for 10 minutes.

[0044] 3. Termination of reaction: After the reaction is completed, immediately add 1.0 mL of DNS reagent and mix well. Heat the test tube in a boiling water bath for 5 minutes and quickly cool to room temperature after sufficient color development.

[0045] 4. Absorbance determination: Measure the absorbance value (A540) of each sample at a wavelength of 540 nm using a spectrophotometer.

[0046] 5. Drawing of standard curve: Use D-galacturonic acid solution of known concentration, treat according to the above method, determine the A540 value, and draw a standard curve.

[0047] 6. Calculation of enzyme activity: Calculate the reducing sugar concentration (μmol) produced by the reaction based on the standard curve. The amount of enzyme required to generate 1 μmol of reducing sugar per minute under the experimental conditions is defined as 1 enzyme activity unit (U).

[0048] The experimental results showed that the enzyme activity of the DS-5 mutant was significantly higher than that of the WT pectinase (see Figure 1 ). The specific data are as follows: WT enzyme: Vmax = 24.92 nmol / min, Km = 0.89 mM.

[0049] DS-5 enzyme: Vmax = 522.74 nmol / min, Km = 7.004 mM.

[0050] The maximum reaction rate of DS-5 was about 21 times that of WT, and the Km value increased, indicating that the affinity of DS-5 for the substrate decreased, but its catalytic efficiency (Vmax / Km) increased overall. This means that under the same conditions, DS-5 can convert pectin substrate into products more quickly.

[0051] Example 4: Functional analysis of mutation sites In order to gain a deeper understanding of the molecular mechanism of enhanced DS-5 enzyme activity, a comprehensive analysis of its 72 amino acid mutation sites was performed.

[0052] 1. Sequence comparison and key site identification: Sequence comparison: The amino acid sequence of DS-5 was compared with that of WT pectinase, and it was found that the mutations were mainly distributed in the surface region of the enzyme and involved non-conserved residues.

[0053] Identification of key sites: Near the active center, the key catalytic residue lysine (K) was found to be replaced by arginine (R). Since both K and R are positively charged basic amino acids, but R has a longer side chain, it may strengthen the charge interaction with the negatively charged pectin substrate.

[0054] 2. Structural analysis and charge distribution: Structural modeling: AlphaFold2 was used to predict the three-dimensional structure of DS-5 and compared with the WT enzyme.

[0055] Potential surface analysis: PyMOL and APBS plugin were used to calculate and visualize the surface charge distribution of the enzyme. The results showed that the positive charge density of DS-5 increased near the active site (see Figure 3 ), which helps to attract negatively charged pectin molecules and increase the probability of substrate-enzyme binding.

[0056] 3. Molecular dynamics simulation: Simulation conditions: Molecular dynamics simulations of the enzyme-substrate complexes of WT and DS-5 were performed for 100 ns using GROMACS software with the same force field parameters and environmental conditions.

[0057] Result analysis: Active site flexibility: DS-5 showed higher conformational flexibility around the active site, which may facilitate the entry of substrates and the release of products, improving the catalytic efficiency of the enzyme.

[0058] Hydrogen bonds and interactions: The number of hydrogen bonds between the enzyme and the substrate was counted, and it was found that the number of hydrogen bonds formed by DS-5 was significantly greater than that of WT, which enhanced the stability of the enzyme-substrate complex.

[0059] Role of key residues: In DS-5, the interactions of residues such as Arg205, Asp226, and Arg260 with substrates are enhanced, and these residues may play an important role in substrate positioning and catalysis.

[0060] Based on the above analysis, the optimization of 72 mutation sites makes the structure of DS-5 more suitable for substrate binding and catalytic reaction. The increased positive charge and higher flexibility around the active site improve the interaction between the enzyme and the pectin substrate and enhance the catalytic efficiency, thus significantly improving the enzyme activity of DS-5.

[0061] Sequence Listing <110> China National Tobacco Corporation Zhengzhou Tobacco Research Institute <120> A high-activity pectinase DS-5 and its preparation and application <160> 1 <211> 324 <212> PRT <213> Pectinase DS-5 <400> 1 GPQGYASLNGGTTGGSGGDTATASTGKEIQDLIDTRSLSSNPNKPLTIYVNGTITPANSDKPLIDVKNHRGSEYPITNISIIGVGTNGEFDGIGIKLSNAHNIIIQNVKIHHVKEGTGTCIEVTDNSKNVWIDHNEFYSDYPGNGDSDYYDGLVDIKRNAEY ITVSWNKFENHWKAMLVGHTDNPALAPDNITYHHNLFKNLNSRVPLIRYANVHMFNNYFEDIKDTAINSRVGARVWVENNYFDNVGSGKKDPVTGEVRGPVGHYYGSSSKGYWNLKGNIFNNTPHSGLKSTTDFEPPYSYTVLSAEEAKAYVLKNSGVGVKT

Claims

1. A highly active pectinase DS-5, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

2.

2. The highly active pectinase DS-5 according to claim 1, characterized in that: Using the deep learning model ProteinMPNN, the wild-type pectinase sequence was redesigned and the highly active mutant DS-5 was screened.

3. The highly active pectinase DS-5 according to claim 2, characterized in that: The redesign specifically includes using the crystal structure of the wild-type pectinase (PDB ID: 3VMW) as input to redesign the pectinase sequence. Based on multiple sequence alignment (MSA), approximately 70% of the highly conserved residues are kept unchanged, and sequence variation design is performed on the remaining approximately 30% of non-conserved residues. The highly active pectinase DS-5 is screened from multiple variant sequences generated by the model.

4. The highly active pectinase DS-5 according to claim 2 or 3, characterized in that: The screening process is to use AlphaFold2 to perform structure prediction and quality assessment on multiple variant sequences generated by the model, and obtain pectinase DS-5 with significantly improved activity through activity identification.

5. The highly active pectinase DS-5 according to claim 1, characterized in that: Compared with the wild-type pectinase (SEQ ID NO: 1), 72 amino acid positions are replaced: the main mutation sites are listed below (sequence position refers to the WT sequence): M8L, A16S, R19D, V20T, E21A, Y22T, A27K, Q28E, Q31D, N35T, R38L, N40S,D43N, E44K, Q56P, G57A, P60D, Q61K, S62P, K72S, A73E, H74Y, E75P, K77T, R96K,S109K, R114K, E117T, A120C, D126N, E140D, F141Y, M156I, T176A, A185P, S186A,K191N, Y198L, N200K, D213N, K222E, N225K, F238W, Q251K, A252K, T255V, F258E,I259V, K260R, W265H, F266Y, P270S, T272K, R278K, V281I, V283N, N287H, H289G,N291K, N295D, T297E, Q303T, Q305L, T308E, Q309E, S312A, S313Y, E315L, Q316K, H317N, I323K, N324T.

6. A method for preparing the highly active pectinase DS-5 according to claim 1, characterized in that: The following steps are involved: 1) Synthesize the DS-5 gene sequence and clone it into an expression vector; 2) Transforming the recombinant vector into host cells for expression; 3) Obtain high-activity DS-5 pectinase.

7. Use of the highly active pectinase DS-5 as claimed in claim 1 in the preparation of juice, jam, textile processing, papermaking or biomass energy conversion.

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